Bambus[4,6]urils as Dual Scaffolds for Multivalent Iminosugar Presentation and Ion Transport: Access to Unprecedented Glycosidase-Directed Anion Caging Agents

Bambusurils, BU[4] and BU[6], were used for the first time as multivalent scaffolds to link glycosidases inhibitors derived from 1-deoxynojirimycin (DNJ). Two linear DNJ ligands having six or nine carbon alkyl azido linkers or a trivalent DNJ dendron were grafted onto octapropargylated BU[4] and dodecapropargylated BU[6] using copper-catalyzed cycloaddition (CuAAC) to yield corresponding neoglycobambus[4] and neoglycobambus[6]urils bearing 8 to 24 iminosugars. The inhibition potencies of neoglycoBU[4], neoglycoBU[6] and neoglycoBU[6] caging anions were evaluated against Jack Bean α-mannosidase and compared to monovalent DNJ derivatives. Strong affinity enhancements per inhibitory head were obtained for the clusters holding trivalent dendrons with inhibitory constants in the nanomolar range (Ki = 24 nM for BU[4] with 24 DNJ units). Interestingly, the anion (bromide or iodide) encapsulated inside the cavity of BU[6] does not modify the inhibition potency of neoglycoBU[6], opening the way to water-soluble glycosidase-directed anion caging agents that may find applications in important fields such as bio(in)organic chemistry or oncology.

To obtain the 12 × 3-valent DNJ-BU [6] conjugate, Br -@propargylBU [6].TBA + 2 was reacted with clickable dendron 14 [38] in the presence of CuSO 4 and sodium ascorbate using microwave irradiations to promote full click coupling (Scheme 3). However, using our general click procedure, only partial functionalization of 2 was observed, affording 6 × 3 valent-DNJ-Tripod-BU [6] 20 with 84% yield. Despite many attempts to optimize the reaction conditions (solvent, stoichiometry, microwave power, temperature, reaction time), it was not possible to obtain fully functionalized BU [6]. We hypothesized that the steric hindrance of the DNJ Tripod 14 did not allow the total functionalization of BU [6] 2. Mass spectra analysis allowed for quantification of the presence of six grafted tripods. The six remaining alkyne functions were observable on the 1 H NMR spectrum of 20.

Glycosidase Inhibition Study
All the new clusters were evaluated against Jack Bean α-mannosidase, a glycosidase highly sensitive to multivalent inhibitor presentation [15][16][17][18][19][20]. The corresponding inhibition constants are summarized in Table 1. The evaluated DNJ-bambusuril conjugates acted as competitive inhibitors, with the notable exception of the series of 12-valent clusters with the shorter C6 linker obtained with the BU [6] scaffold which behaved as mixed inhibitors (Table 1, entries 6-8). The inhibition potency of those new clusters was compared to their corresponding monovalent inhibitors, 21 [22] and 22 [37] (Figure 2), to calculate their relative potency (rp). Dividing the relative potency by the number of active units gives the relative potency per inhitope (rp/n).   As in a previous study with calix [8]arene-based clusters [43], the scaffold has a strong impact on inhibition with the C6 shorter linker. Whereas the inhibitory heads of cluster 3 based on the smaller BU [4] scaffold are individually four times more potent than the reference (rp/n = 4), the ones of clusters 6, 8 and 9 based on BU [6] are less active than the monovalent reference (rp/n < 1) and show a different inhibition mode (Table 1 entries 6-8). Noticeably, the inhibition mode and power of the three BU [6] binding bromide or iodide ions (or with a free cavity) are similar, suggesting an overall similar shape. We previously obtained crystals of Cl − , Br − I − @allyl 12 BU [6].TBA + [12,13] and of Br − , I − @propargyl 12 BU [6].TBA + [14], showing that the halides are included in the cavity and that the geometry of these CH···halide bonds are similar, with distances varying with the halogen ionic radius which thus indicates that the macrocycle retains some flexibility [12][13][14]. Similarly, with C9 linkers, the activities and inhibition mode are not impacted by the presence of anions in the bambusuril cavity (Table 1 entries 9-10). With the longer C9 linker, the impact of the central scaffold, whatever its size, is abolished, giving similar affinity enhancement per DNJ (entries 4, 9 and 10) with C9 enhancement being at least one order of magnitude higher than for the C6 series. This result is in line with previous findings using other scaffolds [15][16][17][18][19][20]43]. The best results were obtained with clusters 5 and 11 grafted with the trivalent dendron with affinity enhancements one order of magnitude higher than those of compounds 4, 7 and 10. The affinity enhancement per inhibitory head of compound 5 is in the same range as for the 24-valent cyclopeptoid bearing the same trivalent dendron [26]. Altogether, those results show that bambusuril scaffolds allow for efficient grafting of glycosidase inhibitors and that this multimerization induces strong affinity enhancements. Moreover, the fact that activity is not influenced by anion binding opens the way to potential therapeutic applications by inducing accumulation of the encaged anion, and potentially radioactive iodide, in a glycosidase rich environment. It is, for example, well-known that β-glucuronidases are present at high concentration in the microenvironment of most solid tumours [44] and that radioactive iodine is used for the treatment of cancers, including thyroid cancers [45][46][47].

General Information and General Experimental Procedures for the Syntheses
Commercially available reagents were used without further purification. All reactions were performed under inert atmosphere using anhydrous solvents which were dried and distilled before being used. Thin-layer chromatograms (TLC) and flash chromatography separations were respectively performed on precoated silica gel 60 F254 plates (0.25 mm) and on Merck Kieselgel 60 (grading 40-63 µm). Microwave syntheses were conducted using a CEM Focused Microwave Discover ® SP-X System reactor. The reactions were performed for 2-4 h under magnetic stirring in 10 or 35 mL sealed Discovered SP vessels closed with Activent®caps. The dynamic control method was used for all microwave reactions where the temperature and the pressure were set (P = 50 W, T = 80 • C, PowerMax on). 1 H NMR spectra (400 MHz) and 13 C NMR spectra (100 MHz) were recorded on a Brucker Avance 400 MHz spectrometer. Chemical shifts and coupling constants are reported in parts per million (ppm) and in Hertz (Hz), respectively. HRMS and electrospray mass spectra (ESIMS) were obtained from an LCT Premier XE using electro spray ionization coupled with a time flight analyzer (ESI-TOF). Infrared spectra (IR) were recorded on a Perkin Elmer UAR Two Spectrum spectrometer. Electrospray mass spectra were obtained using an ESI-Quadripole autopurify Waters (pump: 2545, mass: ZQ2000) mass Spectrometer. Optical rotations were measured on a JASCO P-2000 Polarimeter. Melting points were measured on a Büchi Melting point B540.

General Procedure A for CuAAC Reaction of Propargyl 8 BU[4]
To a solution of octapropargyl bambus [4]

Isothermal Titration Calorimetry Experiments
ITC measurements were performed with a VP-ITC microcalorimeter (Microcal, GE-Healthcare). Experiments were carried out in water and in a solution of K 2 HPO 4 (1.5 mM) in mili-Q water at 298.15 ± 0.1 K. Anion binding to BU was investigated via a classical isothermal titration experiment (10 µL additions) and a single injection method (SIM). Injection of sodium iodide solution was added automatically to the BU solution present in the calorimeter cell while stirring at 307 rpm. Integrated heat effects were analyzed by nonlinear regression using a single-site binding model (Microcal Origin 7). The experimental data fitted to a theorical titration curve, giving the association constant K a , the enthalpy of binding ∆H • and the entropy ∆S • . The free energy ∆G • was calculated from the equation: where T is the absolute temperature. The first smaller addition (2 µL), which was used to compensate for diffusion of the guest from the injector during equilibration, was discarded prior to data fitting.

Inhibition Assays on Jack-Bean α-Mannosidase
p-nitrophenyl-α-D-mannopyranoside and α-mannosidase (EC 3.2.1.24, from Jack Bean) were purchased from Sigma Aldrich, St. Louis, MO, USA. Inhibition constants were determined spectrophotometrically by measuring the residual hydrolytic activities of the α-mannosidase against p-nitrophenyl-α-D-mannopyranoside in the presence and absence of an inhibitor. Each well was filled with a total volume of 100 µL, containing 0.2 M acetate buffer pH 5, inhibitor, substrate and enzyme. All kinetics were performed between 25 • C and 27 • C and started by enzyme addition. After 30-50 min of incubation, the reaction was quenched by addition of 100 µL of 1M Na 2 CO 3 . The absorbance of the resulting solution was determined at 405 nm. K i values were determined in triplicate, using the Dixon and Lineweaver-Burk graphical methods within Microsoft Excel. Stock solutions of inhibitors were prepared with DMSO/buffer for final well DMSO content under 5%. The stability of the enzyme in the presence of the same concentrations of DMSO was controlled and enzyme activity was unaffected.

Conclusions
Propargylated bambus [4,6]urils were fully functionalized by click chemistry with linear N-alkylDNJ ligands either having 6 or 9 carbon alkyl azido linkers or a trivalent DNJ derivative to generate multivalent clusters bearing up to 24 iminosugars. The novelty of this study lies in the unique combination of anion-transporting BU [6] scaffolds with iminosugar inhitopes that have already proven their efficiency towards the multivalent inhibition of glycosidases. For clusters grafted with DNJ ligands linked by short C6 arms, scaffold size showed a strong impact on α-mannosidase inhibition, with the best results obtained for BU [4] clusters. With nonyl chains, the enzyme affinity per inhibitory head increases drastically whichever platform is used, the best affinity increases being obtained with trivalent dendrons providing higher local inhibitor concentration. Bromide and iodide sequestered in BU [6] grafted by DNJ inhibitors were also prepared and their activity was compared to their corresponding anion-free counterparts, showing that the presence of anions neither modified the inhibition potency of the clusters nor their inhibition mode. Our study thus shows a first proof of concept for glycosidase-directed ion caging agents based on unprecedented bambusuril-based iminosugar clusters. Altogether, these results open the way to unique water-soluble probes/therapeutic agents with both strong glycosidase inhibition potency and ion receptor properties.